Fourier transform infrared (FTIR) difference spectroscopy is a powerful probe of structural changes in membrane proteins. It can reveal detailed information about changes in the hydrogen bonding, protonation state and orientation of specific amino acid side chains with microsecond time resolution. However, a general method is needed to assign bands in the infrared difference spectrum to the vibrations of individual amino acid residues in a protein. We propose to do this by combining FTIR difference spectroscopy with site directed isotope labelling (SDIL) of individual atoms in a protein. This approach will provide detailed information about the role of individual amino acid residues in the functioning of a protein. Two bacterial membrane proteins-- bacteriorhodopsin and colicin El have been selected for our initial research. These proteins are representative of a wide range of biomembrane phenomena including energy transduction, active and passive transport, voltage channel gating and signal transduction. A eucaryotic integral membrane protein, the interleukin-8 receptor, has also been selected for FTIR-SDIL studies in the later stages of this project. This protein is representative of the class of seven alpha-helix G-protein coupled receptors. Initial research for this project provides the basis for the engineering ana production of novel tRNAs which will satisfy the requirements for SDIL. Further, we have shown that FTIR difference spectroscopy can probe microgram quantities of protein, detect conformational changes with microsecond resolution and investigate biomembranes in an aqueous environment. In addition to membrane biophysics, the proposed research will have an important impact in other areas including protein folding, enzyme catalysis and the application of 2-D NMR to biological systems.